HBV infection causes acute hepatitis and chronic hepatitis. The majority of individuals who have HBV infection fully recover from the virus infection after 1 to 2 month, but about 10% of them become chronic hepatitis patients. Depending on the age of infection, the rate of becoming chronic infection is much worse, showing over 95% for newborns under the age of 2 months, decreasing as aging progresses, and becoming about 25% for children of age five. Chronic infection of HBV causes increased risk for the development of liver cirrhosis and cancer. HBV surface antigen (HBsAg) is detected in the blood from patients with chronic infection of HBV, however, anti-HBsAg antibody does not appear due to immune tolerance development. Therefore, the final goal for the treatment of chronic HBV infection is to induce anti-HBsAg antibody in serum and remove HBV from the blood and liver.
The therapeutic vaccine described in the present invention is suitable for the treatment of HBV chronic infection, because it breaks immune tolerance and induces immune response against HBV to remove HBV antigen from the blood. Therapeutic vaccines against chronic HBV infection should meet the following requirements; it must be able to break immune tolerance and induce immune responses against HBV, eliciting strong humoral and cell-mediated immunity to resolve the chronic viral infection.
After HBV infection, when polyclonal immune responses were induced, the infection would be resolved. Whereas, when weak oligoclonal immune responses were induced, the infection would lead to chronic infection. This finding suggests that a therapeutic vaccine must contain antigens capable of providing various epitopes needed to induce polyclonal immune responses.
The envelop antigen gene of HBV consists of preS (preS1 and preS2) and S region, and after transcription, three envelop proteins (L protein, M protein and S protein) are synthesized by alternate translation at each of the three initiation codons. L protein consists of about 400 amino acids depending on HBV subtype, comprising the preS1 domain, preS2 domain, and S domain, of which translation initiation starts at preS1 AUG codon. M protein consists of about 281 amino acids, comprising the preS2 domain and S domain, of which translation initiation starts at preS2. S protein consists of about 226 amino acids, comprising only the S domain, and makes up the most abundant portion in the virus particles. The S domains of these envelop proteins are embedded in lipid membrane and form rod-shaped 22 nm and 42 nm particles depending on the ratio of the three different sized envelop proteins. The preS1 and preS2 domains contained in M and L proteins are highly immunogenic and helps to induce S protein specific antibodies in certain congenic mice strains in which the S protein alone is not immunogenic.
As described above, providing the complete set of envelope antigen specific epitopes and HBV antigen as highly immunogenic particle forms is important, developing an HBV vaccine containing entire preS protein and S protein is the aim of present invention. In particular, both animal and human experiments have shown that the vaccine containing both preS and S antigens can induce stronger immune responses and much faster responses than vaccine containing only S antigen. That is, vaccine containing both of preS and S antigens is more immunogenic and induces faster immune responses, as compared to the vaccine containing only S antigen, thereby showing that the L protein is more useful as a therapeutic vaccine for the treatment of chronic hepatitis B.
However, most of commercially available HBV vaccines contain only S protein. This could be due to the fact that expression of the L-protein in particle form seen in HBV chronic patients has not been successful from eukaryotic cell expression systems such as Saccharomyces cerevisiae or Hansenula polymorpha. 
Further, there have been attempts to coexpress a portion of preS antigen (in particular, preS2) and the S antigen in eukaryotic cells such as Saccharomyces cerevisiae or Hansenula polymorpha, however, the products were not found to have sufficient immunogenicity.
In addition, there were other attempts of producing HBV antigen, in which the preS gene was separately expressed to produce the preS antigen only, and then mixed with S antigen. However, the produced linear soluble form of preS antigens was not very immunogenic. Thus, the attempt of improving immunogenicity was unsuccessful in comparison to the entire surface antigen, in which the preS antigen and S antigen are coexpressed in the forms of particles wherein the preS antigens are located at the external surface of particles.
Accordingly, to improve immunogenicity, the preS and S antigens have to be simultaneously expressed, and the preS antigens have to be located on the external surface of particles consisting of S antigens. Recently, Savient Pharma in Israel developed an HBV vaccine, which they claim contains the entire surface antigen, that is, both the preS and S antigens.
The present inventors have successfully developed a CHO cell line that can produce HBV envelop antigen containing preS and S antigens in particle form. They found that, when the entire envelop gene of hepatitis B virus was introduced into a specific vector, all three types of surface proteins (L protein, M protein and S protein) were expressed in the form of particles seen in the blood of HBV chronic patients. Using this antigen, an HBV vaccine comprising the recombinant entire surface antigen has been developed that is highly effective in inducing strong immune responses in transgenic mice and produces HBV antigen in the blood but without any detectable amount of HBV specific antibody.